Together or Apart? Quantifying molecular colocalization in live cell fluorescence microscopyhttp://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1864-0648/homepage/news/22110.en.html
2015-02-09T00:00:00+01:00Toward identification and characterization of molecular interactions in living cells: Quantifying molecular colocalization in live cell fluorescence microscopy.

Bielefeld (Germany)  Interactions between molecules are essential for living organisms. Protein-protein, protein-DNA, and protein-RNA interactions are among the most important regulatory mechanisms in cell biology that determine the fate of cells and are therefore the primary target for pharmaceutical interventions. Accordingly, the quantitative identification and characterization of molecular interactions is of great importance, however, it is one of the most challenging tasks in microscopy.
In living cells, the assessment of interactions between molecular species is typically performed by fluorescent labeling of the interaction partners with spectrally distinct fluorophores and imaging in different color channels. Yet, current methods for determining colocalization of molecules result in outcomes that can vary greatly depending on signal-to-noise ratios, threshold and background levels, or differences in intensity between channels.
A German team led by Thomas Huser (University of Bielefeld) now developed a novel and quantitative method for determining the degree of colocalization in live-cell fluorescence microscopy images for two and even more data channels. Moreover, the new method enables the construction of images that directly classify areas of high colocalization. It is based on close investigation of the Euclidian norm of a vector extracted from a symmetrical correlation matrix.
Starting point was the recently published Correlation-Matrix Method which was initially developed to quantitatively and quickly judge the quality of coincidence events in single-molecule fluorescence experiments. The algorithm analyzes the joint diffusion of two (or more) fluorescent molecules through a confocal excitation volume. This method is based on evaluating the Euclidian length Gamma of a vector derived from a 4 X 4 Hermitian correlation-matrix that contains temporal correlation coefficients calculated from single-molecule diffusion time traces. The essential step in adopting this technique to the analysis of images was the conversion of temporal correlation coefficients to spatial coefficients. An image or a region of interest had to be transcribed into a linear trace of the pixel-to-pixel intensity variation. The Euclidian length Gamma of the vector can then be calculated from the corredponding correlation-matrix.
A novel parameter derived from these calculations and called the Gamma-norm was introduced as a measure for colocalization. For two-color fluorescence experiments this norm adopts values between 0 (i.e. no interactions) and 2 (fully interacting binding partners). Based on this value a direct calculation of the fraction of pixels that do not colocalize or colocalize only randomly is possible.
Compared to other methods, the new correlation-matrix method shows a superior robustness against the influence of varying background or random noise contributions. Even at high noise levels, the Gamma-norm acts as a filter against these factors. An extension to the analysis of three- or multicolor images is straightforward. The technique can readily be applied to 3D and super-resolution microscopy data, as well as data obtained by other contrast methods, e.g. Raman scattering or second harmonic generation (SHG). It could even be expanded to visualize dynamic colocalization effects, i.e. in live-cell movie data.
In order to enable the wider research community to easily test and adopt the Gamma-norm analysis, the team developed a plugin for the widely used open-source image analysis software Fiji, which is available for download (www.physik.uni-bielefeld.de/gica).
(Text contributed by K. Maedefessel-Herrmann)

Neuherberg (Germany)  Optoacoustic imaging, also called photoacoustic imaging, is an imaging technology based on the photoacoustic effect. It enables high-resolution visualization of optical absorbers in biological tissue at depths beyond the operational limits of optical microscopy. The technique is often performed with one-dimensional transducer arrays, in analogy to ultrasound imaging.
Optoacoustic imaging using linear arrays offers ease of implementation but comes with several performance drawbacks, in particular poor elevation resolution, i.e. the resolution along the axis perpendicular to the focal plane. German researchers now propose a bidirectional scanning approach using linear arrays that can improve the imaging performance to quasi-isotropic transverse resolution.
The new approach consists of performing two linear scans of the same region in perpendicular directions, multiplication of the volumes and taking the square root thereof. The team from Technische Universität München and Helmholtz Zentrum München, German Research Center for Environment and Health in Neuherberg, Germany theoretically derived that the proposed method yields a significantly improved resolution in elevation direction with minor losses in lateral resolution and confirmed this behavior in simulation and experiment.
Comparing the novel method with simple addition of the two scans (2lsA mode) showed that taking the product t-norm of the data is the important step for achieving quasi-isotropic resolution. Multiplication improves the resolution because it suppresses those voxels that do not coincide in the reconstructed volumes of the perpendicular scans.
Besides the significant improvement of the transverse resolution when using the proposed reconstruction technique, the noise is suppressed by the multiplication method. Since noise is uncorrelated in independent datasets it is less likely for noise structures to coincide in both datasets of the bi-directional scan. In addition, arc artifacts, a common issue in backprojection algorithms, are reduced. The novel ability to achieve quasi-isotropic resolution in a planar scanning geometry utilizing rather big detector elements might be of great importance in clinical imaging scenarios where SNR is a crucial factor.
(Text contributed by K. Maedefessel-Herrmann)

London (UK)  Fluorescence lifetime imaging (FLIM) of tissue autofluorescence has been shown to provide labelfree contrast between different types and states of tissue. Clinical FLIM can combine lifetime contrast with morphological information to provide a direct comparison between different spatial regions  making it easier to spot differences with respect to normal tissue, e.g. for diagnostic screening and potentially enabling margins of diseased tissue to be identified. To date, however, there have been relatively few clinical FLIM studies, partly due to a lack of suitable instrumentation.
With the aim of applying FLIM to detect and monitor disease in internal organs such as the colon, a British team led by Hugh Sparks (Imperial College London) now developed a flexible wide-field FLIM endoscope based on coherent optical fibre bundles. The device utilizes low average power blue picosecond laser diode excitation sources to induce tissue autofluorescence. Its multimode optical fibre efficiently delivers the excitation radiation to illuminate a 3 mm field of view.
Unfortunately, the multimodal optical fibre propagation necessary to achieve this broad illumination can lead to errors in the lifetime determination due to temporal broadening of the excitation pulses. To address this issue, the researchers characterized the consequent degradation of the spatio-temporal instrument response function (IRF) and incorporated a spatially varying temporal instrument response function in the FLIM analysis.
As prove of principle, the scientists applied their new FLIM endoscope to ex vivo tissue autofluorescence from diseased human larynx biopsies. Using a gain-switched picosecond diode laser operating at 445 nm as the excitation source and an average excitation power of circa 0.5 mW, mm-scale spatial maps of autofluorescence lifetimes could be acquired in about 8 seconds.
To illustrate the instruments potential for FLIM at higher acquisition rates, a higher power mode-locked frequency doubled Ti : Sapphire laser was used to carry out FLIM of ex vivo mouse bowel at up to 2.5 Hz using 10 mW of average excitation power at the specimen.
The results demonstrate the potential of the technique to screen for neoplasia. Based on the flexible wide-field FLIM endoscope, new clinical instrumentation may be developed to aid diagnosis and monitoring of therapeutic interventions.
(Text contributed by K. Maedefessel-Herrmann)

San Diego (CA/USA)  The ability to detect and identify low levels of analytes is of growing importance in medicine and nanotechnology, for example diagnostic monitoring of drug delivery nanoparticle levels, identification of cell-free circulating DNA/RNA and other nanoparticulate biomarkers, and detection of pathogens in clinical and environmental samples. Common wide field epifluorescent microscopes generally lack the resolution and sensitivity to detect low levels of nanoparticulates in biological and clinical samples such as blood and plasma. On the other hand, advanced systems such as confocal microscopes may improve detection. Besides some technical limitations, they are neither low-cost nor easy to use, which is required for point-of-care devices.
The low-cost and widespread penetration of epifluorescent microscopes into laboratory settings, however, makes them a strong candidate for the inclusion in a clinical system designed for low level detection. To put this idea into practice, researchers from the University of California, San Diego (USA) followed a new path: Instead of changing the optical system to better detect an existing sample, they modified the samples appearance to better suit the system by pairing an epifluorescent microscope with a microelectrode array capable of dielectrophoretic (DEP) processes.
Unlike DC electrophoresis, which is limited to the separation of charged particles, DEP is able to separate, concentrate, and trap particles by a variety of properties including size, conductivity, density, permittivity, and charge. Force generated through DEP is dependent upon an induced rather than an intrinsic dipole, so it does not require a particle having a net (nonzero) charge. DEP provides selective concentration and separation of a wide range of particles by frequency modulation.
The combined microscope and DEP microelectrode array system showed to be capable of detecting labeled nanoparticulates at concentrations 50 to 100 times lower than clinically significant levels indicative of pathologic processes. Nanoparticles and DNA biomarkers were rapidly isolated and concentrated onto specific microscopic locations where they could be easily detected by epifluorescent microscopy. In their study, the researchers were able to detect 40 nm nanoparticles down to 23 X 103/µl levels and DNA down to the 200 pg/ml level.
The Californian team is convinced that the synergy of epifluorescent microscopy and DEP microarray devices provides a new paradigm for DNA biomarker diagnostics and the monitoring of drug delivery nanoparticle concentrations.
(Text contributed by K. Maedefessel-Herrmann)

Paris (France)  Membranes play a pivotal role in numerous cell mechanisms, in particular for internalization, adhesion and motility studies. In terms of optical imaging of the membrane, special configurations are needed to remove the light coming from the inner part of the cell. French scientists now show that through-the-objective evanescent microscopy (epi-EM) is a powerful technique to image membranes in living cells.
In label-free evanescent microscopy (EM), configurations similar to total internal reflection fluorescence (TIRF) have been proposed: prism-based or through-the-objective. However, in the latter case, these evanescent techniques have not spread much, with a relative preference for the prism-based configuration also called total internal reflection microscopy (TIRM). The team led by Pierre Bon chose the through-the-objective based configuration (epi-EM), which enabled super-axially resolved tomographic reconstruction of the basal membrane of label-free living cells. The implementation of epi-EM only required an easy to settle illumination/collection scheme on a standard inverted microscope. Only a high-NA objective (NAobj > 1.33) was needed for living biological sample studies and a spatial filter on the epi-illumination arm in order to reject under-critical angle illumination.
Either bead calibration or a multilayer Fresnel model could be used to retrieve nanometric position. Based on a multilayer Fresnel model, the team was able to retrieve the membrane/interface distance with 10 nm precision. The researchers applied this nano-axial tomography to retrieve quantitative information on invagination dynamics of living cell membranes. They studied the membrane elevation map of living cells (Wt HEK-293) during 15 minutes at one frame per second without perturbing the sample.
The results demonstrate that epi-EM gives easily access to axially super-resolved images of unlabeled microscopic samples with almost no microscope modification, and at least a doubled lateral resolution compared to classical TIRM. A study can be of any duration as the signal level is not sensitive to any fluorophore stability dependence and the photoxicity is very low as barely any light is absorbed by the sample. The scientists are convinced that this technique will be useful for cell motility and adhesion studies when the sample cannot be modified (ex. stem cells) or when very fast and/or long studies are required.
(Text contributed by K. Maedefessel-Herrmann)

Florence (Italy)  Many people in the Western World consider it as a social need to hide the effects of aging. For this purpose, different cutaneous rejuvenation treatments have been developed, including a laser-based technique, known as laser resurfacing. Skin irradiation with high-power pulsed laser light induces a thermal shock which stimulates fibroblasts to produce new collagen. This is arranged in a more ordered fashion with respect to the old one, thus reducing wrinkles. To avoid long wound healing time and related risks, an improved method has been developed: micro-ablative fractional laser resurfacing ablates skin only on raster scanned points while the light energy dose deposited is still enough to activate fibroblasts and new collagen production.
The macroscopic effect of such treatment can be measured by naked eye or a white light digital camera. At the microscopic level, it is very difficult to measure collagen modifications at a depth of 0.2 mm or deeper without a biopsy or a similarly invasive technique. Modern imaging techniques provide non-invasive alternatives. A team of Italian scientists from Florence, Fiorentino, and Rome used combined TPF-SHG microscopy to study the effects caused by micro-ablative fractional laser resurfacing.
Two-photon excited fluorescence (TPF) microscopy is a high-resolution laser scanning imaging technique enabling deep optical imaging of tissues. Additional morphological information can be provided by second-harmonic generation (SHG) microscopy, which can be combined with TPF microscopy using the same laser source.
For their study, the researchers performed in vivo two-photon imaging on the forearm of healthy subjects before and forty days after the laser treatment. De novo production of new collagen, as well as an increase in the amount of dermal amorphous component, was found within 40 days from the laser treatment. Visual inspection of the acquired SHG images of dermis by experienced dermatologists demonstrated a stronger collagen synthesis and remodeling on older subjects, whereas the modifications were minimal on younger subjects. The age-dependent effectiveness of the treatment was confirmed by a quantitative spectral analysis, based on the second-harmonic to autofluorescence aging index of dermis (SAAID), which is a recognized scoring method for skin aging assessment by means of non-linear microscopy. In addition, a pattern analysis of SHG images using grey-level co-occurrence matrix (GLCM) was carried out, a well-established method for scoring collagen organization in skin.
While the diagnostic potential of in vivo multiphoton microscopy has already been demonstrated for skin cancer and other skin diseases, this study is the first to demonstrate its potential for a non-invasive follow-up of a laser-based treatment. It also represents a promising tool for the follow-up of other collagen-targeted therapies in dermatology. The researchers are convinced that in the near future non-linear laser scanning microscopy will find a stable place in the clinical dermatological setting.
(Text contributed by K. Maedefessel-Herrmann)

Erlangen (Germany)  Clinical shock is one of the most common causes of the high mortality rate among critically-ill patients in Intensive Care Units. During shock, the transport of oxygenated blood is not sufficient to meet the metabolic demands of the tissue. Early detection is important as shock-preventing medical interventions have to be performed before reversible shock conditions become irreversible.
Although the causes of shock might be different, the basic abnormalities in pathophysiological changes are the same. Early shock can have a variety of signs: a dramatic change in blood oxygenation, specific alteration of the blood pressure and alterations of the blood capillary/vessel network such as period increase, amplitude decrease and diameter changes depending on the type of shock. Hemodynamic monitoring is currently used for shock detection. Unfortunately, the employed techniques provide only a general indication of the patients status and are not always sufficient for determining whether or not a patient is at risk of going into shock. Furthermore, most of these techniques are expensive, difficult to apply, invasive and require complex data processing. For complex and life-threatening infectious diseases with consecutive septic shock development, reliable diagnostics can only be achieved through laborious and time-consuming procedures.
German scientists now propose a novel simple, low-cost method of capillary/vessel spatial pattern monitoring. It analyzes the relative local volume fractions of reduced hemoglobin and oxyhemoglobin concentration from spectrally and spatially resolved diffuse reflectance spectra in order to detect alterations of the spatial distribution of the capillary/vessel spatial network indicating early signs of shock and changes related to pre-shock development.
Using a skin tissue phantom model, the researchers from Erlangen University and University Hospital Erlangen were able to validate their new method. The tissue phantom was made up from epoxy-resin containing capillary channels mimicking the blood capillary/vessel spatial network. Titanium dioxide served as a scatterer and ink as an absorber. The experimental setup included a spatially resolved fiber probe consisting of twenty-nine optical fibers arranged over the skin tissue phantom in an array. Twenty-eight fibers were used as light illumination fibers and one as a detector connected to the spectrometer. The illumination fibers were dynamically switched ON and OFF one after another, starting from the detector side towards the other end.
The preliminary study has shown that this method might be used as a real-time and non-invasive tool for the monitoring of shock development and feedback on the therapeutic intervention for Emergency & Rescue Teams as well as for Intensive Care Units. However, further studies will be needed to confirm the potential clinical utility and accuracy of spectrally-spatially resolved diffuse reflectance techniques in animal and patient models.
(Text contributed by K. Maedefessel-Herrmann)